Early-life inflammation, immune response
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and ageing
Research
Cite this article: Khan I, Agashe D, Rolff J.
2017 Early-life inflammation, immune
response and ageing. Proc. R. Soc. B 284:
20170125.
http://dx.doi.org/10.1098/rspb.2017.0125
Received: 19 January 2017
Accepted: 13 February 2017
Subject Category:
Evolution
Subject Areas:
evolution, immunology
Keywords:
ageing, infection, inflammation,
immunopathology, Malpighian tubules
Authors for correspondence:
Imroze Khan
e-mail: imrozek@ncbs.res.in
Jens Rolff
e-mail: jens.rolff@fu-berlin.de
Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.fig-
share.c.3700150.
Imroze Khan1,2, Deepa Agashe1 and Jens Rolff2
1
National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore
560065, India
2
Freie Universität Berlin, Institute of Biology, Königin-Luise Strasse 1-3, 14195 Berlin, Dahlem, Germany
IK, 0000-0002-0110-2274; DA, 0000-0002-0374-8159; JR, 0000-0002-1529-5409
Age-related diseases are often attributed to immunopathology, which results
in self-damage caused by an inappropriate inflammatory response. Immuno-
pathology associated with early-life inflammation also appears to cause faster
ageing, although we lack direct experimental evidence for this association. To
understand the interactions between ageing, inflammation and immuno-
pathology, we used the mealworm beetle Tenebrio molitor as a study
organism. We hypothesized that phenoloxidase, an important immune effec-
tor in insect defence, may impose substantial immunopathological costs by
causing tissue damage to Malpighian tubules (MTs; functionally equivalent
to the human kidney), in turn accelerating ageing. In support of this hypoth-
esis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in
young adults possibly reduced inflammation-induced autoreactive tissue
damage to MTs, and increased adult lifespan. Our work thus suggests a cau-
sative link between immunopathological costs of early-life inflammation and
faster ageing. We also reasoned that if natural selection weakens with age,
older individuals should display increased immunopathological costs associ-
ated with an immune response. Indeed, we found that while old infected
individuals cleared infection faster than young individuals, possibly they
also displayed exacerbated immunopathological costs (larger decline in MT
function) and higher post-infection mortality. RNAi-mediated knockdown
of PO response partially rescued MTs function in older beetles and resulted
in increased lifespan after infection. Taken together, our data are consistent
with a direct role of immunopathological consequences of immune response
during ageing in insects. Our work is also the first report that highlights the
pervasive role of tissue damage under diverse contexts of ageing and
immune response.
1. Introduction
Immunopathology refers to self-damage caused by an over-reactive immune
system in response to infection, and contributes to the pathology of several
human diseases [1 –3]. In insects, fast-acting but non-specific inflammatory
responses are efficient against invading pathogens, but they also lead to
immunopathology [4]. Cytotoxin-producing effector systems such as the phe-
noloxidase (PO) response pathway can also act against host cells [4– 6].
However, the role of immunopathology in natural populations is still unclear,
with little information on whether immunopathology is a measurable cost of
immune response, with major impacts on fitness. It is important to address
these gaps, because immunopathology owing to inflammatory responses can
have long-term implications. For instance, it was proposed that reduced inflam-
matory exposure during childhood may have contributed significantly to
increased lifespan in modern human industrialized societies [7].
Recently, the impact of an early-life immune response on faster ageing was
studied experimentally in the model insect Tenebrio molitor [8]. The study
suggested that accelerated ageing is caused by immunopathological damage
to Malpighian tubules (MTs). MTs are fluid-secreting epithelia that are
& 2017 The Author(s) Published by the Royal Society. All rights reserved.
 functionally analogous to the human kidney [9,10], playing a
critical role in osmoregulation as well as detoxification of
haemolymph [11]. They are exposed to haemolymph owing
to functional necessities and extend throughout the body
cavity of insects [12]. The lack of an impermeable protective
membrane combined with the spatial disposition makes
MTs particularly vulnerable to damage during an immune
response [13]. However, we lack direct experimental evidence
for the role of damage to MTs as a mediator of faster ageing
owing to early immune response. The PO pathway, a fast-
acting immune effector in insects [14], is a likely candidate
to cause autoreactive tissue damage via cytotoxic interme-
diates in T. molitor [13]. Previous studies have already
established a link between an overactive PO response and
increased mortality in Drosophila melanogaster. For instance,
flies that carried mutations in serpin-encoded spn27A failed
to inhibit overexpression of the PO response, and died faster
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owing to excessive melanization [15]. Also, mutations that
inactivate a signalling serine protease (CG3066) in fruit
flies—required for activation of pro-PO—increased lifespan
after Streptococcus pneumoniae infection [16]. Based on these
results, we propose that the immunopathological damage to
MTs caused by the PO cascade results in accelerated ageing
after early immune response.
In most animals, ageing is associated with a progressive
decline in immune function leading to increased mortality
and morbidity [17 –19]. Yet recent experiments seem to con-
tradict this. For example, in D. melanogaster different
immune genes show age-specific upregulation—older fruit
flies increased the expression of antimicrobial peptides after
infection [20] and harboured lower pathogen load [21].
Similar results were reported for the red flour beetle Tribolium
castaneum, where ageing enhanced multiple components of
immunity such as haemolymph antibacterial activity and
PO response [22]. However, despite this increased immune
response with age, older beetles are more likely to die from
infection. This highlights a mismatch between immune func-
tion and the ability to survive after infection, and questions
whether the observed age-related increase in immunity is
beneficial. Instead, relaxed natural selection in aged individ-
uals [23,24] may result in deregulation of the immune
system, increasing immunopathological costs. We hypoth-
esize that immunopathology associated with such a
deregulated (over-reactive) immune response can directly
contribute to greater post-infection mortality with age.
To begin to understand the complex interplay between
ageing, immune response and immunopathology, we con-
ducted experiments with the holometabolous insect mealworm
beetle T. molitor. We first investigated whether PO-mediated
immunopathological damage to MTs during early-life inflam-
mation resulted in faster ageing. We then tested whether older
individuals experience increased immunopathological risk
and whether this is also mediated by the same mechanisms
as in early-life inflammation.
2. Methods
All experiments were conducted with female T. molitor. The
mealworm beetle is a storage pest of food grain, so it lives in rela-
tively high-density populations in dark places [25]. In our
experiments, we collected experimental beetles from an outbred
stock population maintained at 30 + 28C and supplied with an
ad libitum diet of wheat bran and rat chow, supplemented
with apple every 3 days. Under natural conditions, Tenebrio is 2
exposed regularly to a number of pathogens, including fungi
[26] and microsporidia [27]. However, we have no evidence for
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virulent microbial infections in our stock population. To produce
inflammatory responses without pathogenesis (see below for
experiment 1), we used peptidoglycans (Sigma) derived from
Staphylococcus aureus strain JLA513, a tetracycline resistant
strain known to cause persistent infections in T. molitor [28].
For all live infections (see below for experiments 2 and 3), we
used stationary-phase S. aureus culture (same strain as men-
tioned above). We collected early pupae and determined their
sex by observing the terminal abdominal segments. Upon emer-
gence, only females weighing between 120 and 150 mg were
Proc. R. Soc. B 284: 20170125
retained individually as virgins in grid box containers. We did
not use males in our experiments owing to logistical reasons.
All experimental females were thus controlled for age, mating
status and size. Typically, female Tenebrio beetles attain sexual
maturity and begin to oviposit within a week from adult emer-
gence [29,30]. This applies to the beetle population used in this
study, with more than 97% mating success on day 7 post-eclosion
(I.K. and A. Prakash 2016, unpublished data). We further found
that female beetles undergo reproductive senescence within 42
days post-eclosion (electronic supplementary material, figure
S1), and the mean adult lifespan is 60 days under standard lab-
oratory conditions. Based on these findings, we used 7- and
49-day-old individuals as ‘young’ and ‘old’ adults, respectively.
We subjected both young and old females to experiments on
the same day to enable a direct comparison between age classes.
(a) Experiment 1: impact of immunopathology
associated with early immune activation on ageing
(i) RNAi
To investigate whether reduced immunopathology minimizes
the survival costs of early inflammation, we experimentally
manipulated the degree of immunopathology during early-life
immune response using RNA interference (RNAi) of propheno-
loxidase ( proPO) expression and quantified survival. T. molitor
has at least two proPO genes as documented in Johnston et al.
[31] which likely encode two subunits of a heterodimer.
We used RNAi to knock down the expression of both the
proPO genes in 7-day-old virgin females: a previously sequenced
Tenebrio proPO gene transcript (NCBI accession AB020738.1;
henceforth PO1) [32], and an orthologue to T. castaneum proPO
subunit 1 (henceforth, PO2). We amplified the internal region
of the cDNA sequences encoding PO with T7-tailed primers
(electronic supplementary material, table S1), and used them as
templates to synthesize dsRNA using the T7 MEGAscript kit
(Ambion) according to the manufacturer’s instructions. We
extracted the resulting RNA with phenol/chloroform, resus-
pended it in sterile nuclease-free Ringer solution (128 mM
NaCl, 18 mM CaCl2, 1.3 mM KCl, 2.3 mM NaHCO3 [28]), and
stored it at 2808C until further use. We annealed the comp-
lementary strands by heating at 658C for 30 min and
incubating at 228C for an hour, before injecting 100 ng purified
dsRNA into each beetle. As RNAi control (or early
immune-challenged control), we injected individuals with an
internal region of the cDNA sequence encoding a lysozyme
(Gm-Lys) from Galleria mellonella (Swiss-Prot accession:
P82174), available in our laboratory [33]. Two days after RNAi
manipulation, we injected each beetle with 5 ml peptidoglycan
(concentration: 100 ng in 1 ml of Ringer solution) to deplete the
basal amount of PO in the haemolymph. Finally, after another
2 days, we challenged each beetle with a higher dose of peptido-
glycan (5 ml; 5 mg in 1 ml of Ringer solution) to induce a strong
immune response. Twenty hours later, we quantified RNAi effi-
cacy by performing qPCR (see electronic supplementary
 material, supporting methods for replicate sizes, RNA isolation
and qPCR protocol, and qPCR primers; electronic supplemen-
tary material, table S2). We used the comparative CT method
[34] to estimate relative gene expression (see electronic sup-
plementary material, figure S2 and table S3 for RNAi efficacy).
Following the immune challenge, a subset of beetles from all
RNAi treatments (n ¼ 30 beetles per treatment) was monitored
for total lifespan. Four days after immune challenge, the remain-
ing beetles were either assayed for MT activity (n ¼ 20– 28 beetles
per treatment) or PO response (n ¼ 16 beetles per treatment; see
below for methods). A set of 30 beetles served as unhandled full
controls (control) that remained in the grid box container
throughout the experimental window. In addition, as a pro-
cedural control for the impact of early inflammation on adult
lifespan, 30 beetles received ds-Lys injection (mock RNAi)
followed by a mock immune challenge (with insect Ringer).
Below, we briefly describe the methods for quantifying the
immunopathology and PO responses.
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(ii) Malpighian tubule activity
MT activity is of particular importance in this study owing to its
vulnerability to damage during an immune activation [13].
A previous study used a modified ‘oil drop’ technique [35,36]
to demonstrate a large reduction in MT function owing to immuno-
pathology associated with immune induction [13]. The method
provides an in vitro functional estimate of the ability of isolated
tubules to transport saline across the active cell wall into the
tubule lumen. We estimated the fluid transporting capacity of
MTs harvested from experimental beetles 4 days after immune
challenge, as a proxy for immunopathology owing to immune
response. Each beetle has three pairs (dorsal, lateral and ventral)
of large MTs of varying length but similar secretion rates [37].
Hence, we dissected one tubule from each cold-anaesthetized
animal under cold sterile modified Tenebrio Ringer saline,
prepared as described in Wiehart et al. [38]. We severed the
tubule at the point where it connects to the gut, and removed
another approximately 0.5 mm length from the open end (to
control for the condition of the cut end). Following this, we trans-
ferred a single tubule per beetle to a 60 ml drop of sterile
modified Ringer saline supplemented with 0.05% w/v phenol
red to facilitate visualization, and 0.1 mM l21 dibutyryl cyclic
AMP to stimulate fluid secretion [13,36]. We covered the whole
preparation with mineral oil (Sigma). Next, we pulled the open
end of the tubule out of the saline drop and wrapped it
around 0.1 mm pins (Fine Science Tools) in the mineral oil,
where it secreted fluid. Six hours later, we measured the volume
of the secreted droplet, as well as the length of tubule that
remained within the saline drop using IMAGEJ software. The
volume of the secreted droplet is negatively correlated with the
degree of immunopathological harm to MTs.
(iii) Phenoloxidase response
We measured the PO activity of RNAi-treated beetles by
measuring the rate of formation of dopachrome with a spectro-
photometer [39]. We mixed 2 ml undiluted haemolymph
(collected from a wound between the head and thorax) with
8 ml PBS, and centrifuged the sample at 6500 r.p.m. for 15 min
at 48C. We transferred 5 ml of the supernatant to a 96-well-micro-
plate containing 20 ml PBS and 140 ml distilled water to measure
activated PO enzyme (henceforth, PO activity). We then added
20 ml of L-DOPA substrate into each well, and transferred the
plate immediately into a Microplate reader. We allowed the reac-
tion to proceed at 308C for 60 min, and then measured
absorption at 490 nm once every minute. We quantified PO
enzyme activity as the slope of the linear phase (between 15
and 45 min) of the reaction in each well (Vmax: change in absor-
bance per minute).
(b) Experiment 2: impact of ageing on immune 3
function and post-infection survival
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We grew a S. aureus culture overnight in liquid LB medium to
an OD600 of 95%. We then centrifuged the culture, washed the
pellet three times before re-suspending in insect Ringer solution.
We injected 5 ml of this suspension directly into the haemo-
coel of each individual (approx. 4  106 colony-forming units
(CFUs) per inoculum; see [28]). Control individuals were
injected with 5 ml of Ringer solution. After infection, we redis-
tributed beetles individually into grid boxes under standard
conditions with access to food. For a subset of beetles (n ¼
14 – 15 beetles per age group per treatment), we monitored indi-
Proc. R. Soc. B 284: 20170125
vidual survival daily at 20.00 for 40 days. Another subset was
tested for clearance of S. aureus infection from haemolymph
after 6 h, 1 day, 7 days and 14 days. At each time point, we har-
vested bacterial cells from a group of nine to 11 individuals to
estimate remaining CFUs per beetle using the perfusion bleed-
ing method described by Haine et al. [28]. Briefly, perfused
haemolymph was collected from each beetle and plated on LB
agar containing 5 mg ml21 tetracycline as a selective agent.
The number of colonies observed after 48 h of incubation at
308C should be negatively correlated with the ability to clear
bacterial infection.
Next, we tested whether induced haemolymph antimicrobial
activity against S. aureus cells differed across beetle age groups 1
or 7 days after infection (n ¼ 9 – 13 beetles per infection treatment
per age group), using an in vitro cell killing reaction as described
by Haine et al. [28]. At each time point, we collected 2 ml of
undiluted haemolymph sample as described above, diluted it
with 48 ml PBS and 2 ml of an overnight culture of S. aureus
(approx. 106 CFU), and incubated at 308C with shaking at
150 rpm for 2 h. Following this, we diluted the mixture 800
times and plated out as described above. The number of CFUs
that appeared was counted after 48 h incubation at 308C. The
induced anti-S. aureus activity of the haemolymph was therefore
estimated as the number of S. aureus cells killed during 2 h
of exposure to beetle haemolymph.
Finally, we measured PO response (n ¼ 30 beetles per age
group) and estimated the relative expression of the antimicrobial
peptide-coding genes attacin 2 and tenecin 1 (see electronic sup-
plementary material for qPCR primer sequences and replicate
sizes) as a function of age in naive beetles as described earlier.
Because these assays were performed with uninfected beetles,
they served as an estimate of age-associated changes in base-
line constitutive innate immune function in the absence of
immune induction.
(c) Experiment 3: impact of ageing and infection
on immunopathology
Beetles from both age groups (7 versus 49 days old) were first
infected (or sham-infected) as described earlier (n ¼ 11 – 14 bee-
tles per age group per infection status). Four days later,
we estimated MT function as a proxy for immunopathology
(see experiment 1 for methods). We also manipulated the
impact of bacterial infection on MT activity and tested whether
reducing damage to MTs can rescue the low post-infection survi-
val of older beetles. To this end, we injected 47-day-old beetles
with 5 ml of dsRNA (100 ng ml21) of PO1 or Lys (n ¼ 27 – 34 bee-
tles per RNAi treatment) as described in experiment 1. Two days
later, we infected beetles as described above. We monitored a
subset of individuals for their post-infection survival daily at
20.00 for 25 days (n ¼ 15 –18 beetles per RNAi treatment). The
remaining individuals were tested for MT activity as described
above to quantify immunopathology (n ¼ 12 – 16 beetles per
RNAi treatment).
 (d) Data analysis
Residuals of bacterial clearance data were not normally dis-
tributed (tested with Shapiro – Wilks test). Hence, we log-
transformed the data, and confirmed that the transformed
residuals were normally distributed. Following this, we used
a two-way or one-way ANOVA to test the following effects:
(i) bacterial clearance as a function of age and assay time
(ii) PO response of early immune-challenged beetles as a func-
tion of RNAi treatments. We tested for pairwise differences
between treatments after correcting for multiple comparisons,
using Tukey’s HSD. Non-normally distributed data that could
not be transformed to a normal distribution were analysed
using non-parametric Wilcoxon rank sum tests: (i) MT activity
after early-immune response as a function of RNAi treatments;
(ii) antimicrobial activity as a function of age (analysed separ-
ately for control and infected beetles); (iii) PO response as a
function of age; (iv) MT activity as a function of age and infec-
tion; (v) MT activity of older beetles as a function of RNAi
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treatments. Here, we used a Steel – Dwass test to estimate
pairwise differences.
We used Cox proportional hazard survival analysis to test
the following effects: (i) beetle survival as a function of age
and infection (ii) post-infection survival of old beetles as a func-
tion of RNAi treatments. We did not have any censored values
while analysing the data, as all beetles died within the exper-
imental window. We calculated the impact of treatment (e.g.
bacterial infection or RNAi) as the estimated hazard ratio of
the experimental versus the control group (hazard ratio ¼ rate
of deaths occurring in the experimental group per rate of
deaths occurring in the control group). A hazard ratio signifi-
cantly greater than one indicates an increased risk of mortality
in the experimental group compared with control individuals.
We analysed median and maximum lifespans to measure the
change in ageing rate following early-life immune activation. We
used accelerated failure time (AFT) models [40] to examine the
median lifespan in R using the ‘survival’ package, with each
model separately analysing the difference in median lifespan
between a treatment and unhandled control group. We also com-
pared knockout (PO1 and PO2) versus early immune-challenged
control (EI) beetles to analyse whether PO knockdown increased
lifespan after an early immune response. We found that the Wei-
bull distribution minimized the Akaike’s information criterion
(AIC) value of AFT models, and was thus the most appropriate
method to use for each comparison. For each model, we
estimated the c-parameter (exponential (estimated coefficient
associated with lifespan)) representing the difference in median
lifespan between the two groups, as suggested by Swindell
[40]. A c-parameter value significantly less than 1 indicates
reduction in lifespan and vice versa. For a given comparison,
the value 100(c 2 1) represents the per cent change in median
lifespan of the experimental versus the control group [41].
Maximum lifespan has been suggested as an important
indicator of the ageing process [42]; hence, we used it to test
the impact of early immune response on ageing. We first esti-
mated the 90th percentile lifespan when all treatments were
combined, and then calculated the percentage of beetles in
each treatment group living until this time. We then performed
exact unconditional tests using a contingency table approach to
compare percentage survival of each treatment group with
unhandled control beetles (www.stat.ncsu.edu/exact). We
also compared knockout beetles versus immune-challenged
control beetles (i.e. PO1 versus EI or PO2 versus EI) to test
whether PO knockdown increased maximum lifespan signifi-
cantly. We used a binomial, two-way model to generate a
pooled z-score test p-value for each comparison. To obtain
the treatment effect for each comparison, we divided the per-
centage survival of the respective control groups by that of
the experimental group.
3. Results 4
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(a) Phenoloxidase response after early inflammation
increases mortality and organ damage
We found that an early immune challenge in young adults
caused an increase in PO response (figure 1a and electronic
supplementary material, table S4a), with a concomitant
reduction in MT activity (figure 1b and electronic supplemen-
tary material, table S4b). We further observed that RNAi
knockdown of the PO response reduced MT damage in
immune-challenged beetles, resulting in a limited reduction
Proc. R. Soc. B 284: 20170125
of fluid secretion rate (compare figure 1a,b and electronic sup-
plementary material, table S4a,b). We note that if difficulty in
MT excretion alters haemolymph pH in immune-challenged
beetles, PO enzyme kinetics could also change without
actual changes in proPO expression. This explanation can
be ruled out, however, as the RNAi manipulation reduced
PO transcripts which strongly correlates with the decreased
PO enzymatic activity determined by the standard spectro-
photometric method (compare electronic supplementary
material, figure S2 and figure 1a).
An AFT model showed that early immune-challenged
beetles also had shorter lifespans compared with full control
and procedural control beetles (figure 2a,b and electronic sup-
plementary material, table S5a). We did not detect mortality
until 16 days following the immune challenge, suggesting
that early immune challenge did not have an immediate
impact on survival (figure 2a). The c-parameter was lowest
in immune-challenged control (EI) beetles, suggesting a
reduction in median lifespan ( per cent decline with respect
to full control, 100(c 2 1): approx. 35%) following an early-
life immune response (figure 2b and electronic supplementary
material, table S5a). The negative impact of an early immune
challenge on beetle lifespan could also be reversed by RNA
interference of proPO transcripts (e.g. PO1 and PO2) in
immune-challenged beetles (figure 2a,b and electronic sup-
plementary material, table S5a). RNAi of both the
transcripts extended the lifespan by approximately 31–32%
compared with immune-challenged control (EI) beetles
(figure 2b and electronic supplementary material, table S5a).
Analysis of maximum lifespan produced similar results as
that of median lifespan. The 90th percentile of overall survival
time was 74 days (pooling all individuals across treatments);
the percentage of immune-challenged individuals surviving
to this time was significantly lower than control groups
(EI ¼ 3%, PO1 ¼ 10%, PO2 ¼ 10%, PC ¼ 23%, control ¼ 27%;
also see figure 2c and electronic supplementary material,
table S5b for treatment effects). These data suggest accelera-
tion in ageing owing to early-life immune response
(compare 90th percentile survival for each experimental
group: FC ¼ 79.5; PC ¼ 83.1, EI ¼ 57.6; PO1 ¼ 73.8, PO2 ¼
73.8). Finally, we found that RNAi knockdown of the PO
response significantly increased maximum lifespan compared
with immune-challenged EI beetles with normal PO levels,
suggesting delayed ageing in knockout groups (compare
ageing acceleration in figure 2c and electronic supplementary
material, table S5b).
(b) Immune responses increase with age
We found a rapid clearance of bacterial cells in both young
and old beetles: more than 98% cells were removed within
 (a) table S6c) and higher expression of the antimicrobial peptides 5
0.030
B attacin 2 and tenecin 1 (figure 3d and electronic supplemen-

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0.025 tary material, table S3), even in the absence of a previous
phenoloxidase activity

bacterial infection.
Vmax (OD min–1)

0.020 C
0.015 C (c) Older beetles die faster after bacterial infection
A To test if an age-related increase in immunity also confers
0.010
greater survival benefits, we tested the impact of S. aureus
0.005 infection on the survival of young versus old beetles. Com-
pared with young beetles, we found that old beetles died
0 n much faster after an infection (hazard ratio: sham-infection
ol

rly PO1 tion

O2 ion

Proc. R. Soc. B 284: 20170125

versus infection in old beetles ¼ 12.59, p , 0.001; sham-infec-
tio
ntr

i-P mat
ma

i- ma
co

tion versus infection in young beetles ¼ 5.18, p , 0.001;

lam

NA am

NA am
figure 3e and electronic supplementary material, table S6d).
+ R infl

+ R infl
inf
rly

rly

In contrast, beetle age did not alter the survival of

ea

ea

ea

sham-infected beetles significantly (figure 3e and electronic

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(b) supplementary material, table S6d).

0.030
A
vol. secreted (µl h–1 mm–1)
Malpighian tubule activity

0.025
(d) Increased immune response impairs Malpighian
0.020 tubule activity via immunopathology
0.015 We found that ageing itself reduced the baseline MT activity
in sham-infected beetles. Bacterial infection impaired the
0.010 fluid transport ability of MTs in both age groups, but the rela-
C C
tive impact of infection was much larger in infected older
0.005 B
beetles (approx. 95% reduction, compared with an approx.
0 73% reduction in young beetles; figure 3f and electronic
supplementary material, table S7a). As observed for early
n
ol

rly PO1 ion

O2 ion
tio
ntr

i- mat

i-P mat
ma

immune-challenged beetles, we found that RNAi-mediated

co

lam

NA am

NA am

knockdown of pro-PO1 partially rescued the MT activity of

+ R infl

+ R infl
inf

infected older beetles (figure 3g and electronic supplementary

rly

rly
ea

ea

ea

material, table S7b), and proPO1 knockdown beetles survived

treatment
Figure 1. Impact of RNAi-mediated knockdown of prophenoloxidase tran-
scripts on (a) phenoloxidase (PO) response (n ¼ 16 beetles per treatment)
and (b) Malpighian tubule (MT) activity (n ¼ 20–28 beetles per treatment),
serving as a proxy for immunopathological damage, measured at day 4 post-
immune challenge (i.e. day 15 post-emergence). PO activity was measured as
Vmax of the enzymatic reaction with L-DOPA substrate. MT activity was
measured as the rate of fluid secretion. Significantly different groups are indi-
cated by distinct alphabets (based on Tukey’s HSD/Steel–Dwass test).
Control ¼ unhandled control beetles; early inflammation ¼ early immune
challenged control beetles; early inflammation þ RNAi-PO1 ¼ RNAi of PO1
transcript followed by an early inflammation; early inflammation þ RNAi-
PO2 ¼ RNAi of PO2 transcript followed by an early inflammation.
6 h after infection. However, older individuals showed
consistently lower bacterial loads across different time
points (i.e. 6 h, 24 h, 7 days and 14 days) after infection
(figure 3a and electronic supplementary material, table S6a).
Next, we tested the ability of cell-free haemolymph to
kill S. aureus cells 1 or 7 days after infection with live bacteria
(or sham infection). We found that haemolymph from
older infected beetles showed a significantly higher antibac-
terial response compared with younger beetles (figure 3b
and electronic supplementary material, table S6b). In contrast,
age did not have a strong influence on the antibacterial
activity of cell-free haemolymph harvested from sham-
infected beetles (electronic supplementary material, figure S3
and table S6b). Older beetles also showed an enhanced PO
response (figure 3c and electronic supplementary material,
longer after infection than wild-type beetles (hazard ratio:
knockout versus infected wild-type beetles ¼ 0.396; p ¼ 0.01;
figure 3h and electronic supplementary material, table S7c).
We thus suggest that bacterial infection induced immune
upregulation in older beetles, but also incurred greater immuno-
pathological damage to MTs, resulting in greater mortality
despite higher immune function.
4. Discussion
In the work presented here, we provide evidence that is con-
sistent with an important role of immunopathological costs in
the diverse contexts of ageing and immune response in the
model insect T. molitor. We first show that early-life inflam-
mation leads to faster ageing. Early-life immune activation,
without the direct cost of a live and replicating pathogen,
was sufficient to reduce the activity of MTs, a vital insect
organ that is functionally equivalent to the vertebrate
kidney. Subsequently, we demonstrated that experimental
suppression of the PO response partially rescued MT activity
and increased survival. These results highlight that greater
collateral damage to vital organs by PO is possibly one of
the mechanisms that cause accelerated death of older beetles
after early-life inflammation. Although our work focuses on
an insect system, it may provide the first empirical support
for the proposed link between childhood inflammation,
immunopathology and reduced human lifespan [7]. Next,
we describe the immunopathological risk associated with
an ageing immune system and its impact on the lifespan of
older individuals. While ageing increased several aspects of
 (a) 6
1.0

rspb.royalsocietypublishing.org
proportion surviving
control
0.8
sham inflammation
0.6
early inflammation
0.4 early inflammation + RNAi-PO1
0.2 early inflammation + RNAi-PO2
0
0 20 40 60 80
lifespan after early inflammation (in days)

Proc. R. Soc. B 284: 20170125

(b) (c)
reduction in median longevity (c)

10
1.2 A A B

ageing acceleration
8
C C
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1.0 6

0.8 B 4 C C
2 A A
0.6
0
+ inf 1 n

+ inf 1 n
l

RN nfla tion

O tion

RN fla tion

O tion
n

n
tro

tro
rly PO tio

rly PO tio
io

io
n

n
ea Ai- ma

ea i- ma
at

at
a

i-P a

i-P a
co

co
m

A mm

A mm
m

m
2

2
m

m
m

m
m

m
fla

RN la

fla

RN la
fla

fla

in
in

in

A
in

in
i
rly

rly
rly

rly
am

am
ea

ea
ea

ea
+

+
sh

sh

treatment treatment
Figure 2. (a) Survival curves of adults after an early immune challenge that induced inflammation (n ¼ 30 beetles per treatment). (b) Impact of phenoloxidase
(PO) knockdown on median lifespan. Survival of each experimental group was compared with the unhandled control group using an accelerated failure time model.
The c-parameter denotes the effect of the immune challenge treatment on survival, averaged over total survival time. Error bars represent 95% confidence intervals.
(c) Impact of PO knockdown on maximum lifespan. ‘Ageing acceleration’ on the y-axis denotes the reduction in maximum lifespan caused by immune challenge
treatments (the percentage of survivors to the 90th percentile survival time in the full control group/the percentage of survivors to the 90th percentile survival time
in the experimental group). Control ¼ unhandled control beetles; sham inflammation ¼ procedural control for early life immune challenge; early inflammation ¼
early life immune challenged control beetles; early inflammation þ RNAi-PO1 ¼ RNAi of PO1 transcript followed by an early inflammation; early inflammation þ
RNAi-PO2 ¼ RNAi of PO2 transcript followed by an early inflammation.

beetle immunity such as PO response, antimicrobial peptide that immunopathology is indeed the most probable
gene expression, induction of haemolymph antibacterial explanation for our observed results.
activity and the ability to clear S. aureus infection, it also In a prior study, Ayres & Schneider [16] had predicted a
severely impaired MTs and compromised the ability to direct role for the PO response pathway in immunopathologi-
survive after bacterial infection. We found that RNAi knock- cal damage and increased post-infection mortality of fruit
down of the PO response once again minimized damage to flies. Our data not only support this prediction, but also indi-
MTs of old beetles, partially rescued fluid transport ability cate impaired MT activity as an important mechanism
and extended adult lifespan after infection. Thus, one of the underlying the immunopathological consequences of the
most interesting implications of our work is the likely role PO response and associated reduction in lifespan. We note
of immunopathological damage to MTs that underlies that other components of the immune system can also be
the effects of early immune activation as well as the later involved in immunopathology, and the PO pathway may
impacts of infection. Because we used fluid secretion rate as not be the sole determinant of immunopathology. For
a proxy for immunopathological damage and given that instance, the tumour necrosis factor-like protein encoded by
fluid transportation can be energy-intensive, a possible the gene eiger is known to cause immunopathology in fruit
alternative is that the reduced fluid transport after immune flies [43]. NF-kB signalling, which is involved in chronic
challenge indicates a trade-off between immune induc- inflammation in vertebrates [44], may also cause immuno-
tion and MT excretion [9]. Yet another possibility is that pathology in insects, though it is poorly studied. We thus
immune activation may alter the phosphorylation state of suggest the need for further studies to test the fitness impacts
dFOXO of the tubules, which may in turn change its fluid of immunopathology caused by other immune effectors.
transport ability without direct immunopathological conse- Another important finding of our study is a mechanism
quences [9]. In both cases, a general immune activation to explain the discordance between immune response and
should lead to altered MT fluid secretion. However, previous the host fitness post-infection, reported in this study as well
work in Tenebrio shows that MT function decreased with local as in flour beetles [22] and fruitflies [45,46]. Such a mismatch
rather than with general immune activation [13], suggesting suggests that the inherent ability to mount an immune
 (a) (b) 7

activity no. cells killled × 104

haemolymph anti-S. aureus
6 105
Y

rspb.royalsocietypublishing.org
Y
5 A AA O O

(mean log (CFU))

A 100 B

bacterial load
4 B
A A
B A
3 95
A
2 B
90
1
0 85
6h day 1 day 7 day 14 day 1 day 7
time of assay time of assay
(c) (d)

Proc. R. Soc. B 284: 20170125

PO response (OD min–1 × 10–3)

expression relative to rpl27a

8 0.06
Y
0.05 O B
6 B
0.04
A
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4 0.03
0.02
2 B
0.01
A A
0 0
young old attacin tenecin 1
age gene of interest
(e) (f)
0.030
vol. secreted (ml h–1 mm–1)
proportion of live animals

Page = 0.01, Ptreatment < 0.001 sham infection

1.0 A
0.025 infection
0.8
0.020
0.6
0.015
0.4 B
YSI 0.010 B
0.2
YI
0 OSI 0.005
OI C
0
0 10 20 30 40 young old
lifespan after infection (days) age
(g) (h)
proportional of live animals

0.030
vol. secreted (ml h–1 mm–1)

Ptreatment < 0.001

0.025 1.0
A 0.8
0.020
0.6
0.015
0.4
0.010 0.2
C SI
0.005 0 I
B RNAi-I
0
SI I RNAi-I 0 10 20 30 40
treatment lifespan after infection (days)
Figure 3. (a – f ) Impact of age on (a) bacterial clearance at different time points after infection (n ¼ 9 –11 beetles per age group per treatment per time point);
(b) haemolymph anti-S. aureus activity of infected beetles at different time points (n ¼ 9 – 13 beetles per age group per treatment per time point); (c) pheno-
loxidase (PO) response of naive beetles (n ¼ 30 beetles per age group); (d ) expression of antimicrobial peptide genes attacin and tenecin-1 in naive beetles relative
to an internal control (rpl27a) (n ¼ 5– 6 pairs per age group per gene); (e) post-infection survival (n ¼ 14 – 15 beetles per age group per treatment) and (f )
Malpighian tubule (MT) activity after bacterial infection (n ¼ 11 – 14 beetles per age-group per infection status); (g,h) Effect of RNA interference of PO1 transcript
on (g) immunopathological damage to MTs (n ¼ 12 – 16 beetles per RNAi treatment) and (h) post-infection survival in older beetles (n ¼ 15 – 18 beetles per RNAi
treatment). Bacterial clearance was measured as log10 of colony-forming units (CFUs) recovered from perfused haemolymph after injection of S. aureus cells into each
beetle. Haemolymph antibacterial activity was measured as the number of S. aureus cells killed during 2 h of exposure to beetle haemolymph. PO response and
immunopathology were measured as described in figure 1. Significantly different groups are indicated by distinct alphabets (based on Tukey’s HSD/Steel –Dwass
test). For panels (a,b,d), alphabet assignments are meaningful only within each time point (or gene) ( partitioned by dashed vertical lines), and are not comparable
across time points (or genes). Y ¼ young beetles; O ¼ old beetles; YSI ¼ young sham-infected beetles; OSI ¼ old sham-infected beetles; YI ¼ young infected
beetles; OI ¼ old infected beetles; PC ¼ sham infection with insect ringer; I ¼ S. aureus infection; RNAi þ I ¼ RNAi followed by S. aureus infection.

response is not a reliable predictor of host fitness after infec- older individuals. We find that bacterial infection rendered
tion. Our data suggest that these contradictory results may be MTs of older beetles almost dysfunctional (approx. 95%
mediated via a large reduction in MT activity in infected reduction). This implies that older infected beetles should
 have difficulty in maintaining the water balance and accumu-
late toxic waste products inside the haemocoel [47]—a
condition analogous to potential uraemic poisoning. We thus
propose that older beetles succumb to infection faster than
younger beetles owing to the inability of damaged MTs to
effectively maintain physiological homeostasis. This may
also suggest that older beetles pay greater immunopathologi-
cal costs of increased immune activation compared with
young beetles. In fact, several studies in vertebrates show
that ageing leads to chronic inflammatory responses via
maladaptive impacts of the innate immune system, contribut-
ing to the pathology of several age-related illnesses [19,48].
An exaggerated inflammatory response with age can induce
lethal immunopathology in mice: old individuals die faster
owing to hepatocyte necrosis caused by an elevated level of
interleukin-17 and neutrophil activation [44]. Another study
found increased post-infection mortality in older mice
Downloaded from https://royalsocietypublishing.org/ on 05 December 2021
with suppressed anti-inflammatory cytokine interleukin-10
expression, suggesting a link between overactive inflamma-
tory responses and increased risk of mortality [49]. We thus
conclude that the observed increase in beetle immunity
with age actually represents immune activation at an
unnecessarily high level without any adaptive value. Instead,
ageing leads to a dramatic increase in the negative impacts of
inflammation in beetles, and the net impact of the immune
response compromises old individuals’ fitness post-infection.
Moreover, ageing may also reduce tolerance to infection, the
ability to limit negative impacts of pathogens, or self-damage
(discussed in reference [50]). Consequently, older individuals
may increasingly rely on direct immune activation to limit
pathogen burden, which could rapidly escalate the immuno-
pathological risk. However, we stress that currently there is
no evidence for this hypothesis in T. molitor.
Our work also highlights the intrinsic aspects of physiologi-
cal ageing in insects. We find that multiple components of
immunity (e.g. PO and antimicrobial peptides) increase as a
function of age, even in the absence of pathogenic infection
(in naive beetles). A plausible explanation is that the observed
increase in baseline constitutive innate immune responses is a
by-product of general relaxation of gene regulation associated
with ageing (see reference [23]). We further speculate that the
strength of selection on old individuals may be too weak [23]
to effectively prevent such misregulation of the immune
system, resulting in increased risk of immunopathology.
Indeed, in support of the above prediction, our data reveal a
baseline reduction in MT functioning with age in naive beetles.
This perhaps indicates an age-dependent trade-off between
investment in immune function and prevention of its immuno-
pathological costs. It is possible that such increases in immune
responses and immunopathological damage to vital organs
(even without infection) could be a key feature of senescence.
However, we lack direct empirical evidence for this hypothesis.
Further manipulative experiments are thus necessary to test
whether experimental suppression of inflammatory pathways
References
1. Bach JF. 2002 The Effect of infections on 2.
susceptibility to autoimmune and allergic diseases.
N. Engl. J. Med. 347, 911 –920. (doi:10.1056/
NEJMra020100)
(e.g. RNAi of PO response in insects) results in reduced 8
organ damage in older uninfected individuals, in turn,
rspb.royalsocietypublishing.org
improving their lifespan.
We note an alternative possibility where the observed
age-specific differences in survival and MT function could
be artefacts of using only virgin beetles. In some insects,
virgin females also develop ovaries on par with their mated
counterparts but refrain from laying eggs [51]. If storing
unfertilized eggs bears additional costs, then it is possible
that the physiological burden of carrying eggs increased
with age, resulting in observed physiological defects.
However, there is currently no evidence for such costs of
Proc. R. Soc. B 284: 20170125
egg retention in T. molitor or any related coleopterans (but
see blood-feeding insects [52]). Furthermore, in previous
work with T. castaneum flour beetles, we found that older
individuals increased immunity and yet, die faster after infec-
tion, regardless of their mating status [22]. Hence, we do not
expect that the physiological burden of carrying unfertilized
eggs should impact our experimental results.
Overall, our work has important implications for the evol-
ution of maladaptive immune pathology in natural
populations. Natural selection might be almost blind to the
self-damage associated with the immune response, because
the costs are usually paid at a later stage in life [23]. From a
public health perspective, such relaxed selection can pose a
serious problem to an ageing human population, potentially
exacerbating the autoimmune disease crisis worldwide
[1,53,54]. We suggest that intimate connections between
immune action and immunopathology may drive these hall-
marks of ageing. We hope that our observations encourage
further empirical work for a deeper understanding of life-
history trade-offs and fitness impacts associated with
immune function and its immunopathological outcome.
These are necessary to understand how and why apparently
maladaptive immunopathological features of an immune
system have evolved.
Data accessibility. Data available from the Dryad digital repository:
http://dx.doi.org/10.5061/dryad.dk034 [55].
Authors’ contributions. I.K. and J.R. conceived of and designed exper-
iments; I.K. carried out experiments and analysed data; I.K. and
J.R. wrote the manuscript. D.A. helped draft the manuscript. All
authors gave final approval for publication.
Competing interests. We have no competing interests.
Funding. We acknowledge funding and support from the Centre for
International Collaboration, Free University of Berlin; DAAD and
SERB-DST Young Investigator Grant supplements to I.K.; the
National Centre for Biological Sciences, India; a DST Inspire Faculty
fellowship to D.A.; and a European Research Council (EVORESIN)
grant supplement to J.R.
Acknowledgements. We are grateful to Paul Johnston, Dino McMahon,
Caroline Zanchi, Saurabh Mahajan and Vrinda Ravi Kumar for feed-
back on the manuscript. We thank Jayjit Das, Caroline Zanchi, Arun
Prakash and Dipendra Nath Basu for their help during experiments
and data analysis.
Dye C, Scheele S, Dolin P, Pathania V, Raviglione M. 3. Bekker LG, Moreira AL, Bergtold A, Freeman S,
1999 Global burden of tuberculosis: estimated Ryffel B, Kaplan G. 2000 Immunopathologic effects
incidence, prevalence, and mortality by country. of tumor necrosis factor alpha in murine
JAMA 282, 677– 686. (doi:10.1001/jama.282.7.677) mycobacterial infection are dose dependent. Infect.
 Immun. 68, 6954– 6961. (doi:10.1128/IAI.68.12.
6954-6961.2000)
4. Urabe K, Aroca P, Tsukamoto K, Mascagna D,
Palumbo A, Prota G, Hearing VJ. 1994 The inherent
cytotoxicity of melanin precursors: a revision.
Biochim. Biophys. Acta - Mol. Cell Res. 1221,
272–278. (doi:10.1016/0167-4889(94)90250-X)
5. Nappi AJ, Vass E, Frey F, Carton Y. 1995 Superoxide
anion generation in Drosophila during melanotic
encapsulation of parasites. Eur. J. Cell Biol. 68,
450–456.
6. Zhao P, Lu Z, Strand M, Jiang H. 2008 Antiviral,
antiparasitic, and cytotoxic effects of 5,6-
dihydroxyindole (DHI), a reactive compound
generated by phenoloxidase during insect immune
response. Insect Biochem. Mol. Biol. 141, 520–529.
(doi:10.1016/j.ibmb.2011.04.006)
Downloaded from https://royalsocietypublishing.org/ on 05 December 2021
7. Finch CE, Crimmins EM. 2004 Inflammatory
exposure and historical changes in human life-
spans. Science 305, 1736 –1739. (doi:10.1126/
science.1092556)
8. Pursall ER, Rolff J. 2011 Immune responses
accelerate ageing: proof-of-principle in an insect
model. PLoS ONE 6, e19972. (doi:10.1371/journal.
pone.0019972)
9. Davies SA, Overend G, Sebastian S, Cundall M,
Cabrero P, Dow JAT, Terhzaz S. 2012 Immune and
stress response ‘cross-talk’ in the Drosophila
Malpighian tubule. J. Insect Physiol. 58, 488–497.
(doi:10.1016/j.jinsphys.2012.01.008)
10. Pacheco CA, Ravazi A, de Azeredo Oliveria MTV,
Alevi K. 2014 Review: malpighian tubule, an
essential organ for insects. Entomol. Ornithol.
Herpetol. Curr. Res. 3, 2– 4. (doi:10.4172/2161-
0983.1000122)
11. McGettigan J et al. 2005 Insect renal tubules
constitute a cell-autonomous immune system that
protects the organism against bacterial infection.
Insect Biochem. Mol. Biol. 35, 741–754. (doi:10.
1016/j.ibmb.2005.02.017)
12. Chapman RF. 1998 The insects: structure and
function. Cambridge, UK: University of Cambridge
Press.
13. Sadd BM, Siva-Jothy MT. 2006 Self-harm caused by
an insect’s innate immunity. Proc. R. Soc. B 273,
2571–2574. (doi:10.1098/rspb.2006.3574)
14. Cerenius L, Lee BL, Söderhäll K. 2008 The proPO-
system: pros and cons for its role in invertebrate
immunity. Trends Immunol. 29, 263–271. (doi:10.
1016/j.it.2008.02.009)
15. De Gregorio E et al. 2002 An immune-responsive
Serpin regulates the melanization cascade in
Drosophila. Dev. Cell 3, 581–592. (doi:10.1016/
S1534-5807(02)00267-8)
16. Ayres JS, Schneider DS. 2008 A signalling protease
required for melanization in Drosophila affects
resistance and tolerance of infections. PLoS Biol. 6,
e305. (doi:10.1371/journal.pbio.0060305)
17. Deveale B, Brummel T, Seroude L. 2004 Immunity
and aging: the enemy within? Aging Cell 3,
195–208. (doi:10.1111/j.1474-9728.2004.00106.x)
18. Shanley DP, Aw D, Manley NR, Palmer DB. 2009 An
evolutionary perspective on the mechanisms of
immunosenescence. Trends Immunol. 30, 374–381.
(doi:10.1016/j.it.2009.05.001)
19. Licastro F, Candore G, Lio D, Porcellini E, Colonna-
Romano G, Franceschi C, Caruso C. 2005 Innate
immunity and inflammation in ageing: a key for
understanding age-related diseases. Immun. Ageing
2, 8. (doi:10.1186/1742-4933-2-8)
20. Zerofsky M, Harel E, Silverman N, Tatar M. 2005
Aging of the innate immune response in Drosophila
melanogaster. Aging Cell 4, 103 –108. (doi:10.1111/
j.1474-9728.2005.00147.x)
21. Khan I, Prasad NG. 2013 The aging of the immune
response in Drosophila melanogaster. J. Gerontol.
A. Biol. Sci. Med. Sci. 68, 129–135. (doi:10.1093/
gerona/gls144)
22. Khan I, Prakash A, Agashe D. 2015
Immunosenescence and the ability to survive
bacterial infection in the red flour beetle Tribolium
castaneum. J. Anim. Ecol. 85, 291–301. (doi:10.
1111/1365-2656.12433)
23. Hamilton WD. 1966 The moulding of senescence by
natural selection. J. Theor. Biol. 12, 12– 45. (doi:10.
1016/0022-5193(66)90184-6)
24. Mueller LD, Rose MR. 1996 Evolutionary theory
predicts late-life mortality plateaus. Proc. Natl Acad.
Sci. USA 93, 15 249 –15 253. (doi:10.1073/pnas.
93.26.15249)
25. Ramos-Elorduy J, González EA, Hernández AR, Pino
JM. 2002 Use of Tenebrio molitor (Coleoptera:
Tenebrionidae) to recycle organic wastes and as
feed for broiler chickens. J. Econ. Entomol. 95,
214 –220. (doi:10.1603/0022-0493-95.1.214)
26. Meyling NV, Eilenberg J. 2007 Ecology of the
entomopathogenic fungi Beauveria bassiana and
Metarhizium anisopliae in temperate
agroecosystems: potential for conservation biological
control. Biol. Control 43, 145–155. (doi:10.1016/j.
biocontrol.2007.07.007)
27. Armitage SAO, Siva-Jothy MT. 2005 Immune
function responds to selection for cuticular colour in
Tenebrio molitor. Heredity 94, 650 –656. (doi:10.
1038/sj.hdy.6800675)
28. Haine E, Moret Y, Siva-jothy M, Rolff J. 2008
Antimicrobial defence and persistent infection in
insects. Science 322, 1257–1259. (doi:10.1126/
science.1165265)
29. Tschinkel W, Willson C, Bern HA. 1967 Sex
pheromone of the mealworm beetle. (Tenebrio
molitor). J. Exp. Zool. 164, 81 –85. (doi:10.1002/jez.
1401640108)
30. Morales-Ramos JA, Rojas MG, Kay S, Shapiro-Lian
DI, Tedders WL. 2012 Impact of adult
weight, density, and age on reproduction of
Tenebrio molitor (coleoptera: Tenebrionidae).
J. Entomol. Sci. 47, 208 –220. (doi:10.18474/0749-
8004-47.3.208)
31. Johnston PR, Makarova O, Rolff J. 2014 Inducible
defenses stay up late: temporal patterns of immune
gene expression in Tenebrio molitor. G3 4,
947 –955. (doi:10.1534/g3.113.008516)
32. Dobson AJ, Johnston PR, Vilcinskas A, Rolff J. 2012
Identification of immunological expressed sequence
tags in the mealworm beetle Tenebrio molitor.
J. Insect Physiol. 58, 1556–1561. (doi:10.1016/j. 9
jinsphys.2012.09.009)
rspb.royalsocietypublishing.org
33. Johnston PR, Rolff J. 2015 Host and symbiont jointly
control gut microbiota during complete
metamorphosis. PLoS Pathog. 11, 1 –11. (doi:10.
1371/journal.ppat.1005246)
34. Schmittgen TD, Livak KJ. 2008 Analyzing real-time
PCR data by the comparative CT method. Nat.
Protoc. 3, 1101–1108. (doi:10.1038/nprot.2008.73)
35. Maddrell SH, Overton JA. 1990 Transport in insect
Malpighian tubules. Methods Enzymol. 192,
617–632. (doi:10.1016/0076-6879(90)92099-Y)
Proc. R. Soc. B 284: 20170125
36. Neufeld DS, Leader JP. 1998 Cold inhibition of
cell volume regulation during the freezing of
insect Malpighian tubules. J. Exp. Biol. 201,
2195– 2204.
37. Nicolson S. 1992 Excretory function in Tenebrio
molitor: fast tubular secretion in a vapour-absorbing
insect. J. Insect Physiol. 38, 139–146. (doi:10.1016/
0022-1910(92)90043-D)
38. Wiehart UIM, Nicolson SW, Eigenheer RA, Schooley
DA. 2002 Antagonistic control of fluid secretion by
the Malpighian tubules of Tenebrio molitor: effects
of diuretic and antidiuretic peptides and their
second messengers. J. Exp. Biol. 205, 493 –501.
39. Zanchi C, Troussard J, Martinaud G. 2011 Differential
expression and costs between maternally and
paternally derived immune priming for offspring in
an insect. J. Anim. Ecol. 80, 1174–1183. (doi:10.
1111/j.1365-2656.2011.01872.x)
40. Swindell WR. 2010 Accelerated failure time models
provide a useful statistical framework for ageing
research. Exp. Gerontol. 44, 190–200. (doi:10.1016/
j.exger.2008.10.005)
41. Patel K, Kay R, Rowell L. 2006 Comparing
proportional hazards and accelerated failure time
models: an application in influenza. Pharm. Stat. 5,
213–224. (doi:10.1002/pst.213)
42. Wang C, Li Q, Redden DT, Weindruch R, Allison DB.
2004 Statistical methods for testing effects on
‘maximum lifespan’. Mech. Ageing Dev. 125,
629–632. (doi:10.1016/j.mad.2004.07.003)
43. Brandt SM, Dionne MS, Khush RS, Pham LN, Vigdal
TJ, Schneider DS. 2004 Secreted bacterial effectors
and host-produced eiger/TNF drive death in a
Salmonella-infected fruit fly. PLoS Biol. 2, e418.
(doi:10.1371/journal.pbio.0020418)
44. Stout-Delgado HW, Du W, Shirali AC, Booth CJ,
Goldstein DR. 2009 Aging promotes neutrophil-
induced mortality by augmenting IL-17 production
during viral infection. Cell Host Microbe 6,
446–456. (doi:10.1016/j.chom.2009.09.011)
45. Corby-Harris V, Habel KE, Ali FG, Promislow DEL.
2007 Alternative measures of response to
Pseudomonas aeruginosa infection in Drosophila
melanogaster. J. Evol. Biol. 20, 526–533. (doi:10.
1111/j.1420-9101.2006.01267.x)
46. Ramsden S, Cheung YY, Seroude L. 2008 Functional
analysis of the Drosophila immune response during
aging. Aging Cell 7, 225 –236. (doi:10.1111/j.1474-
9726.2008.00370.x)
47. Maddrell SH, Overton JA. 1988 Stimulation of
sodium transport and fluid secretion by ouabain in
 an insect Malpighian tubule. J. Exp. Biol. 137,
265–276.
48. Vasto S et al. 2007 Inflammatory networks in
ageing, age-related diseases and longevity. Mech.
Ageing Dev. 128, 83 –91. (doi:10.1016/j.mad.2006.
11.015)
49. Belloni V, Faivre B, Guerreiro R, Arnoux E, Bellenger
J, Sorci G. 2010 Suppressing an anti-inflammatory
cytokine reveals a strong age-dependent survival
cost in mice. PLoS ONE 5, e12940. (doi:10.1371/
journal.pone.0012940)
Downloaded from https://royalsocietypublishing.org/ on 05 December 2021
50. Medzhitov R, Schneider DS, Soares MP. 2012
Disease tolerance as a defense strategy. Science
335, 936 –941. (doi:10.1126/science.1214935)
51. Shukla S, Shilpa MC, Gadagkar R. 2013 Virgin wasps
develop ovaries on par with mated females, but lay
fewer eggs. Insect. Soc. 60, 345– 350. (doi:10.1007/
s00040-013-0299-1)
52. Xue R, Ali A, Barnard DR. 2005 Effects of forced
egg-retention in Aedes albopictus on adult survival
and reproduction following application of DEET as
an oviposition deterrent. J. Vector Ecol. 30, 45 –48.
53. Zaccone P, Fehervari Z, Phillips JM, Dunne DW, 10
Cooke A. 2006 Parasitic worms and inflammatory
rspb.royalsocietypublishing.org
diseases. Parasite Immunol. 28, 515–523. (doi:10.
1111/j.1365-3024.2006.00879.x)
54. Finch CE. 2010 Evolution of the human lifespan and
diseases of aging: roles of infection, inflammation,
and nutrition. Proc. Natl Acad. Sci. USA 107,
1718– 1724. (doi:10.1073/pnas.0909606106)
55. Khan I, Agashe D, Rolff J. 2016 Data from: Early
inflammation, immunopathology and aging. Dryad
Digital Repository. (doi:10.5061/dryad.dk034)
Proc. R. Soc. B 284: 20170125